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目的构建表达大鼠促红细胞生成素(erythropoietin,EPO)的真核表达质粒pEGFP-N1/EPO,并检测其在体外的表达。方法从大鼠肾脏获得EPO基因片段并用RT-PCR方法扩增,将其连接于真核表达质粒pEGFP-N1,测序鉴定后,用脂质体包裹转染大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs),采用免疫组化和RT-PCR检测EPO的表达。结果经酶切和测序鉴定证实本实验构建的重组表达质粒正确,该质粒在体外转染大鼠MSCs细胞后可表达EPO蛋白。结论成功构建的pEGFP-N1/EPO重组质粒能在体外表达EPO蛋白。
Objective To construct the eukaryotic expression plasmid pEGFP-N1 / EPO expressing rat erythropoietin (EPO) and test its expression in vitro. Methods EPO gene fragment was obtained from rat kidney and amplified by RT-PCR. The fragment was ligated to the eukaryotic expression plasmid pEGFP-N1. After sequencing, the gene fragment was transfected into rat mesenchymal stem cells cells, MSCs). The expression of EPO was detected by immunohistochemistry and RT-PCR. Results After digestion and sequencing, we confirmed that the constructed recombinant plasmid was correct. The plasmid can express EPO protein in vitro after transfected into rat MSCs. Conclusion The recombinant plasmid pEGFP-N1 / EPO can express EPO protein in vitro.